Hair follicles are exceptional organs that repeatedly self-regenerate in a mature body throughout nearly the entire lifetime. Elucidation of the mechanism of this self-regeneration is expected to lead to clinical applications for which there are considerable needs, including hair loss therapy by tissue and cell transplant, and the construction of natural and functionally superior hair sheets containing hair follicles and sebaceous glands. Accompanying the growing interest in stem cell research in recent years, research on follicular epithelial stem cells (epidermal cells) has progressed rapidly, and the properties of hair dermal papilla cell serving as mesenchymal cells specific to hair follicles have gradually been determined. Hair dermal papilla cell fulfill the role of a so-called control tower, sending out activating signals to follicular epithelial stem cells for self-regenerating hair follicles, and have been determined to be indispensable along with follicular epithelial stem cells in evaluation of follicle reconstitution (Kishimoto et al., Proc. Natl. Acad. Sci. USA (1999); Vol. 96, pp. 7336-7341).
Hair follicle reconstitution experiments in animal models have been conducted using various methods for the purpose of hair follicle regeneration. Weinberg et al., J. Invest. Dermatol. (1993), Vol. 100, pp. 229-236 describes a hair follicle reconstitution method using a cell transplant method. The transplant system of Weinberg et al. has a complex constitution consisting of hair dermal papilla cell, newborn animal epidermal cells (including follicular epithelial stem cells) as well as the addition of mouse 3T3 cells. According to the method of Weinberg et al., hair follicles are regenerated without adding newborn animal epidermal cells containing follicular epithelial stem cells to the transplant system. However, this is thought to be phenomenon that occurred due to the difficulty in completely removing undifferentiated epidermal cells (follicular epithelial stem cells) and primordial hair follicles from the dermal cell fraction. Subsequently, Kishimoto et al. (op. cit.) succeeded for the first time in isolating and purifying hair dermal papilla cell, and as a result of conducting a hair follicle reconstitution experiment according to a cell transplant method in an animal model using the isolated and purified hair dermal papilla cell, hair follicles were reorganized and hair growth was observed when a cell fraction containing a combination of hair dermal papilla cell and epidermal cells was transplanted. However, it was found that hair growth is not observed when a cell fraction that does contain either hair dermal papilla cell or epidermal cells is transplanted.
The hair dermal papilla cell purification method according to Kishimoto et al. utilizes the fact that hair dermal papilla cell have the property of specifically expressing versicans (chondroitin sulfate proteoglycans). However, it carries out isolation and concentration by using the expression of versicans by a transgenic mouse model produced using DNA in which a reporter gene is linked to a versican gene as an indicator. Thus, this method requires the production of a transgenic mouse and purification of hair dermal papilla cell by a cell sorter. A large amount of hair dermal papilla cell are required to actually regenerate hair follicles and cause hair growth in hair follicle reconstitution methods in particular (for example, 5 million cells per transplant).
Consequently, this method requires the production of a large number of transgenic mice and the long-term use of a high-speed cell sorter, thereby resulting economic problems as well as problems in terms of work time and labor. Although the isolation of hair dermal papilla cell is also described in, for example, Prouty et al., American J. Pathol. (1996) Vol. 148, No. 6, pp. 1871-1885, this method has a complex procedure due to repeated fractionation by centrifugal separation, has a low level of purity and generates a low yield.
Thus, in previous methods for purifying hair dermal papilla cell, it was difficult to acquire an amount of isolated and purified active hair dermal papilla cell sufficient for transplant, for example, and the role of active hair dermal papilla cell in the regeneration of hair follicles was unable to be completely elucidated. In particular, it has been virtually impossible with purification methods of the prior art to determine the suitable ratio of hair dermal papilla cell and epidermal cells in follicle reconstitution systems for regenerating hair follicles.